Inhibition Of Arsenic Trioxide In Bcr-Abl Mutant Cells And The Mechanism

Chronic myeloid leukemia is kind of clonal malignant myeloproliferative disorder of the hematopoietic stem cells, characterized by the presence of the abnormal Ph+ chromosome. The incidence of the disease is moving up, which is reported to be 1/100000 in the western and 3.6/100000 in China, taking up the third place in adult leukemia. The incidence of chronic myeloid leukemia worldwide accounts for 15 percent of adult leukemia.Imatinib is the most effective tyrosine kinase inhibitor to cure chronic myeloid leukemia at chronic phase. The clinical application of IM for the treatment of CML has opened up a new way. Althougth Imatinib has made most CML patients auquiring more durable cytogenetic response, part of patients may relapse after auquiring effective response, which is a more obvious phenomenon in CML at accelerate phase, blast phase and Ph+patients. The point mutant of Abl kinase doman is the primary cause of the failure to cure CML patients with Imatinib and replase of CML.At first, this experiment investigated the effect of Imatinib on the growth inhibition of all Bcr-Abl mutant cell lines in vitro by trypan blue exclusion. The results showed that wild-type cells were sensitive to IM, while the other cells with Bcr-Abl point mutations had a certain resistance to IM, which were IM resistant cell lines. Therefore, to look for other effective therapeutic drugs is a imperative task in clinical work.Having achieved great success in healing acute promyelocytic leukemia, Arsenic Ttioxide, as a multi-target medicine has also made great progress in healing other types of leukemia and solid tumors. This experiment attempted to investigate the effect of arsenic trioxide (ATO) on the growth inhibition of Bcr-Abl mutant cell lines including T315I mutant in vitro and its potential mechanism. The growth inhibition of ATO on Bcr-Abl wild type and IM-resistant cell lines were measured by trypan blue exclusion. ATO induced cell growth inhibition and apoptosis in both Bcr-Abl wild and IM-resistant cells in a dose dependent manner. However, ATO showed stronger growth inhibition on Bcr-Abl point mutant cells compared with the wild type cells. The IC50 of ATO in mutation cells was 2.04-4.63 fold lower than that in wild type cells. However, IC50 of ATO in K562 and K562R cells was not different, the latter showed no any mutation.95 percent of hematopoietic stem cells in CML patients contain Bcr-Abl fusion gene, and Bcr-Abl chimeric protein locate in the central part of complex signaling network, excessive tyrosine kinase activity of the Bcr-Abl protein makes itself and its downstream signaling pathway over-activation. Bcr-Abl mRNA levels were measured by fluorescence quantitative PCR after treatment Bcr-Abl wild cells and IM-resistant cell lines with ATO, and Bcr-Abl protein expression levels were detected by Western blot. Results showed that Bcr-Abl mRNA levels were unchanged and the protein expression were decresed with the increase of ATO concertrations in two cell types. In views of the important role of Bcr-Abl chimeric protein in CML pathogenesis, we considered that ATO reduced Bcr-Abl protein may be one of the reasons of the special mechanism of IM resistant cell lines.There was some sensitivity difference between Bcr-Abl wild type and IM-resistant cell lines after treatment with ATO. In order to study the causes of this difference, Glutathione (GSH) levels were analyzed using Glutathione Assay Kit. The GSH levels in Bcr-Abl point mutant cells were lower than that in wild type cells, but quite similar in K562 and K562R cells. Apoptosis was assayed by AnnexinV & PI staining after treatment of these cells with glutathione biosynthesis inhibitors BSO. The results showed that the growth inhibition of Bcr-Abl point mutant and wild type cells by ATO treatment was significantly enhanced. The growth inhibition of Bcr-Abl point mutant cells by ATO was more significant than wild type cells, which may be related to the content of intracellular GSH. But we do not know what causes intracellular GSH decrease in the mutation cell lines and this will be our next focus.